Not applicable.
Over the centuries, the bubonic plague (also known as the Black Death) has claimed the lives of millions of people. The disease is characterized by chills, fever, vomiting, diarrhea, painful swollen lymph nodes (buboes), blackening of the skin caused by ruptured blood vessels, and a very high mortality rate (up to 75% if left untreated). Treatment with antibiotics in the early stages of the infection is generally effective.
Bubonic plague is caused by the bacterium Yersinia pestis, which is transmitted to humans from rats or other rodents by fleas that feed on infected rodents and then bite humans. Reservoirs of the bacteria persist today, and attempts to eliminate wild rodent plague have proven ineffective. Occasional outbreaks of the deadly disease continue to occur, particularly in small towns, villages, and rural areas in developing countries.
While bacteria carry genetic material in their chromosomes, bacteria also often carry genetic material in loops of DNA called plasmids. Bacterial plasmids are nonessential, extrachromosomal genetic elements capable of autonomous replication. The genetic material in plasmids often encodes functions required for maintenance of the plasmid in its bacterial host and sometimes encodes optional functions that promote survival of the bacterial host under certain environmental conditions. Pathogenicity determinants are commonly plasmid-encoded, and fall within the category of optional plasmid-encoded functions.
Yersinia pestis is a facultative intracellular parasite which harbors at least three different plasmids, designated pCD1, pPCP1, and pMT1, which are necessary for full virulence of the organism. One of the plasmids, designated pCD1, is also found in the enteropathogenic species Yersinia pseudotuberculosis and Yersinia enterocolitica (Ferber, et al. Infect. Immun. 31:839-841, 1981; Portnoy, et al. Curr. Topics Microbiol. Immunol. 118:29-51, 1985), whereas pMT1 and pPCP1 are unique to Y. pestis (Brubaker. Clinical Microbiol Rev. 4:309-324, 1991). Plasmids pMT1 and pPCP1 are thought to promote deep tissue penetration by Y. pestis and to contribute to the acute infection associated with this species. The Y. pestis genome shares much homology with that of Y. pseudotuberculosis, (Bercovier, et al. Curr. Microbiol. 4:225-229, 1980; Moore, et al. Inter. J. Sys. Bacteriol. 25:336-339, 1975), yet the infection caused by the latter organism is usually mild and self limiting (Butler, Plague and other yersinia infections, p. 111-159. In W. B. Greenbugh III and T. C. Merigan (eds.), Current topics in infectious disease, Plenum Press, New York, N.Y., 1983). 
An understanding of the differences in the pathogenesis of Y. pestis and Y. pseudotuberculosis may be afforded by comparing polynucleotide sequences or genes found on pMT1 or pPCP1 plasmids, and which are unique to Y. pestis. It has been found that Y. pestis strains lacking the pCD1 plasmid are completely avirulent. Therefore, determination of the complete pCD1 sequence may provide important information about the role of the plasmid in virulence in various pathogenic yersiniae.
The 9.5 kb plasmid pPCP1 encodes a bacteriocin termed pesticin, a pesticin immunity protein and a plasminogen activator activity. Loss of this plasmid increases the LD50 of the organism by a factor of one hundred thousand, as measured by subcutaneous injection in the mouse model. (Sodeinde, et al. Science 258:1004-1007, 1992). 
The second plasmid unique to Yersinia pestis, designated pMT1, is a 100 kb plasmid that encodes the capsular protein Fraction 1 and the murine toxin (Protsenko, et al. Genetika 19:1081-1090, 1983). The genes for the capsular proteins have been cloned and sequenced using Y. pestis strain EV76 (Galyov, et al. FEBS Lett. 277:230-232, 1990; Galyov, et al. 286:79-82, 199.1; Karlyshev et al. FEBS Lett. 305:37-40, 1992). The role of these proteins in plague pathogenesis has not been unequivocally determined, and the effect of mutational loss of these proteins on the LD50 varies, depending on the animal model and route of infection (Brubaker Curr. Top. Microbiol. 57:111-118, 1972; Brubaker Rev. Infect. Diis. 5: S748-S758, 1983). However, pMT1 does appear to contribute to the acute phase of plague infection, as evidenced by a reduced morbidity associated with infection by strains lacking pMT1 (Drozdov, et al. J. Med. Microbiol. 42:264-268, 1995; Samoilova, et al. J. Med. Microbiol. 45:44.0-444, 1996;Welkos, et al. Contrib. Microbiol. Immunol. 13:229-305, 1995). 
Information pertaining to the genetic characterization of the pMT1 molecule is limited. The size of the plasmid has been found to vary, either from variations in the versions of the plasmids or in technique to measure the plasmids, from 90 kb to 288 kb (Filippov, et al. FEMS Microbiol. Lett. 67:45-48, 1990). It is known that pMT1 is an integrative plasmid capable of integrating into Y. pestis chromosome with high frequency and at multiple sites, with integration likely resulting from IS100 homology between the plasmid and chromosome (Protsenko, et al. Microbiol. Pathogen 11:123-128, 1991).
Previous characterization of pMT1 has identified five genes that may be involved in the synthesis of murine toxin (MT) and F1 capsule antigen, both known virulence factors. Expression of both the capsular protein and murine toxin genes has been characterized with respect to environmental cues (e.g., temperature and-calcium) (Du, et al. Contrib. Microbial. Immunol. 13:321-324, 1995). F1 capsule synthesis is maximal at 37xc2x0 C. in the absence of extracellular calcium, conditions similar to those that induce expression of a major Y. pestis virulence determinant (Straley Rev. Infect. Dis. 10:S323-S326, 1988; Straley Microbial. Pathoaen 10:87-89, 1991; Straley et al. Proc. Natl. Acad. Sci. USA 78:1224-1228, 1981). Murine toxin expression is induced at 26xc2x0 C., conditions similar to those that would be expected to occur in the flea vector. The occurrence of plasmid genes that are induced under widely different conditions suggests regulation of Y. pestis virulence determinant expression by at least two networks.
The plasmid pCD1 is found in Y. pestis, as well as in certain other pathogenic Yersinia species, including Y. pseudotuberculosis and Y. enterocolitica. The plasmid encodes a complex virulence property called the low-Ca2+ response (LCR). The LCR was discovered in Y. pestis growing in vitro, where the bacteria respond to the absence of Ca2+ at 37xc2x0 C. by the strong expression and secretion of a virulence protein called V antigen, or LcrV. In certain media, expression of LcrV is accompanied by a response termed xe2x80x9crestriction,xe2x80x9d in which the yersiniae undergo an orderly metabolic shutdown and cease growth. Under LCR-inductive conditions, the transcription, translation, and secretion of a set of virulence proteins called Yops (for Yersinia outer proteins) is maximally induced. The operons encoding these and other similarly regulated operons on the LCR plasmid have been referred to as the LCR stimulon (LCRS). Millimolar concentrations of Ca2+ permit full growth at 37xc2x0 C., reduced expression of LcrV and Yops, and essentially no secretion of these proteins. Under ambient temperature conditions outside a mammalian host, the Yops and LcrV proteins are produced at a low, basal level and are not secreted, which suggests that the LCR is designed to function within a mammal. Expression of LCR is apparently modulated by other environmental factors, including Mg2+, Clxe2x88x92, Na+, glutamate, nucleotides, and anaerobiosity. The molecular basis for these effects has not been determined, but these elements of environmental modulation could be important in adjusting virulence protein expression and secretion in response to the wide range of niches that yersiniae are expected to encounter during an infection.
The pCD1 plasmid also encodes a type III secretion system called Ysc (for Yop secretion) that is involved in the secretion of Yops, LcrV, and some regulatory proteins in the LCR. The Ysc system is locally activated by cell contact at the interface between a bacterium and eukaryotic cell. This cell to cell contact causes the opening of the secretion system""s inner and outer gates (LcrG and LcrE (or YopN), respectively), thereby allowing secretion of negative regulatory proteins (e.g., LcrQ also called YscM, a key regulatory protein). Secretion of negative regulatory proteins allows full transcriptional activation of LCRS operons by LcrF, an AraC-like activator protein.
Yops are secreted locally, without processing. The secretion mechanism recognizes two signals: one in the first 45 nucleotides of the yop mRNA and one related to a domain that has been found for some Yops to bind a specific Yop chaperone (Syc), also encoded by the LCR plasmid Certain of the Yops (e.g., YopB, YopD, YopK) are involved in targeting effector Yops (YopE, YopH, YpkA, YopM, and possibly YopJ) into the eukaryotic cell. Once inside the cell, the effector Yops act on intracellular target molecules, thereby interfering with cellular signaling and cytoskeletal functions. LcrV acts functions both as a regulatory protein involved in Yop secretion and targeting and as a potent anti-host protein. LcrV is the only LCRS protein that is secreted in large amounts into the surrounding medium by yersiniae in contact with eukaryotic cells. LcrV adversely affects the host organism when administered alone to mice, whereas all other secreted proteins depend on the Ysc machinery of yersiniae, in intimate contact with mammalian cells, for delivery into the mammalian cells.
Expression of the LCR has a profound immunosuppressive effect that results from the interference with innate defenses at the site of infection and the host organism""s inability to mobilize an effective cell-mediated immune response. Y. pestis, and, in immunocompromised individuals, the enteropathogenic yersiniae grow unchecked in the lymphoid system in a fulminant-disease associated with high mortality, absent appropriate antibiotic treatment. In contrast, yersiniae lacking the LCR plasmid pCD1 are completely avirulent.
Several other important pathogens have virulence systems with many striking similarities to the LCR; however, the LCR is the best characterized of these and remains a prototype for investigations at the forefront of molecular pathogenesis.
A more complete understanding of the role of LCR plasmids may be obtained by determining the entire sequence of an LCR plasmid.
The development of additional sequence information from plasmids of Y. pestis is needed for comprehensive efforts in the detection, diagnosis, prophylaxis and treatment of infections caused by the organism.
One aspect of the present invention is an isolated Yersinia pestis plasmid pMT1- or pCD1-specific polynucleotide sequence selected from the group consisting of any portions of the sequences present in SEQ ID NO:1 through SEQ ID NO:6 set forth below.
The present invention is in part summarized by the presentation of the complete nucleotide sequence of two plasmids from Yersinis pestis, which enables diagnostic, prophylactic and therapeutic tools to be developed for use in combating the pathogen.
The DNA sequences of the present invention may include an open reading frame (ORF), an insertion sequence element, or a plasmid maintenance function, for example.
It is an object of the invention to provide essentially the entire sequence of pMT1 and pCD1 from Yersinia pestis KIM5 to allow methods of detecting, diagnosing, preventing, and treating infections with Yersinia pestis.